CN113156132B - Preparation method of chip with antifouling microgel, chip and sensor - Google Patents
Preparation method of chip with antifouling microgel, chip and sensor Download PDFInfo
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- CN113156132B CN113156132B CN202110338792.3A CN202110338792A CN113156132B CN 113156132 B CN113156132 B CN 113156132B CN 202110338792 A CN202110338792 A CN 202110338792A CN 113156132 B CN113156132 B CN 113156132B
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- 230000003373 anti-fouling effect Effects 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 70
- 239000013078 crystal Substances 0.000 claims abstract description 49
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000000243 solution Substances 0.000 claims abstract description 41
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000012153 distilled water Substances 0.000 claims abstract description 33
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 21
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000011259 mixed solution Substances 0.000 claims abstract description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims abstract description 11
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims abstract description 9
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims abstract description 9
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims abstract description 9
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000004473 Threonine Substances 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 8
- 239000000203 mixture Substances 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 8
- 238000011010 flushing procedure Methods 0.000 claims abstract description 7
- 238000003825 pressing Methods 0.000 claims abstract description 7
- HSGLYHHOCKTAEL-UHFFFAOYSA-N 1-(6,6-dimethoxycyclohexa-2,4-dien-1-yl)ethanone Chemical compound COC1(OC)C=CC=CC1C(C)=O HSGLYHHOCKTAEL-UHFFFAOYSA-N 0.000 claims abstract description 6
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims abstract description 6
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims abstract description 6
- ULVXDHIJOKEBMW-UHFFFAOYSA-N [3-(prop-2-enoylamino)phenyl]boronic acid Chemical compound OB(O)C1=CC=CC(NC(=O)C=C)=C1 ULVXDHIJOKEBMW-UHFFFAOYSA-N 0.000 claims abstract description 6
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims abstract description 6
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims abstract description 6
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000001105 regulatory effect Effects 0.000 claims abstract description 6
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 3
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 3
- 230000001678 irradiating effect Effects 0.000 claims abstract 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 241000252506 Characiformes Species 0.000 claims description 10
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 5
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 235000011837 pasties Nutrition 0.000 claims description 5
- 239000010453 quartz Substances 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 238000000861 blow drying Methods 0.000 claims description 3
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 18
- 108090000623 proteins and genes Proteins 0.000 abstract description 18
- 238000012360 testing method Methods 0.000 abstract description 9
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 abstract 1
- 238000004140 cleaning Methods 0.000 abstract 1
- 230000000379 polymerizing effect Effects 0.000 abstract 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 36
- 239000008103 glucose Substances 0.000 description 36
- 210000003296 saliva Anatomy 0.000 description 21
- 238000001514 detection method Methods 0.000 description 11
- 238000010382 chemical cross-linking Methods 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 238000003380 quartz crystal microbalance Methods 0.000 description 7
- 239000008280 blood Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000010408 film Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010340 saliva test Methods 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000004043 responsiveness Effects 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000001055 chewing effect Effects 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000011898 label-free detection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/56—Acrylamide; Methacrylamide
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F265/00—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00
- C08F265/10—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00 on to polymers of amides or imides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
- C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
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- Polymers & Plastics (AREA)
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Abstract
The invention provides a preparation method of an antifouling microgel chip, which comprises the following steps: synthesis of microgel: adding 3-acrylamidophenylboronic acid, acrylamide, glycidyl methacrylate, N, N' -methylenebisacrylamide and azodiisobutyronitrile into acetonitrile, polymerizing at 90 ℃ and repeatedly cleaning with distilled water; synthetic microgel antifouling layer: threonine and serine in a molar ratio of 1:1, adding the mixture into an ethanol solution, regulating the pH value, dispersing microgel into the mixed solution, washing the product by distilled water, adding ethanol, and centrifuging; is fixed on the crystal oscillator piece: and dispersing the microgel anti-fouling layer, N-vinyl pyrrolidone, ethylene glycol dimethacrylate and 2, 2-dimethoxy-phenyl ethanone in dimethyl sulfoxide to prepare a prepolymerization solution, placing the crystal oscillator sheet with the front surface facing downwards on the prepolymerization solution, pressing the back surface of the crystal oscillator sheet, irradiating for 1 hour by an ultraviolet lamp, flushing with distilled water and drying with nitrogen. The invention can eliminate inaccurate testing caused by high non-specificity of protein.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to a preparation method of an antifouling microgel chip, the chip and a sensor.
Background
Diagnosis and treatment of diabetes requires periodic or (ideally) continuous control of glucose levels in the blood in order to optimize treatment and improve the quality of life of the diabetic patient. Currently, blood glucose level detection mainly relies on two methods, one is to puncture a finger to measure blood glucose and the other is to insert a microprobe into the skin to continuously measure glucose in intercellular fluid. However, both methods are invasive methods. In order to meet the medical requirements of diabetics and improve the life quality of the diabetics, noninvasive technology has become a research hotspot for clinical and scientific research. In particular, the correlation between the concentration of glucose in saliva and the blood glucose level is high, and saliva has the natural advantages of safety, convenience for real-time acquisition and the like, so that the noninvasive blood glucose monitoring based on saliva becomes a research hot spot.
The glucose content in saliva is very low, about 1/100 to 1/50 of the blood glucose. Therefore, a high requirement is placed on a saliva glucose meter. Quartz Crystal Microbalance (QCM) is a mature mass sensitive tool for detecting biomolecules, which can detect sub-nanometer mass changes. Compared with other electronic components, the QCM has the characteristics of high sensitivity, high response speed, low production cost, strong real-time measurement capability, simple integration and the like, and becomes a suitable tool for detecting various molecular recognition events such as liquid DNA, nucleic acid, protein, bacteria and the like on line. In general, polymer coatings or biochemically modified QCM chips form thin films with specific biomolecule-recognition properties, which enable label-free detection of biomolecules exposed to biological fluids. Along with the absorption of target molecules by the QCM chip, the oscillation frequency of the chip is reduced, and the on-site real-time monitoring can be performed. However, the extremely low content of glucose molecules in saliva (3.6-36 mg/L) makes the frequency change very small, and worse, the decrease of the oscillation frequency of the crystal oscillator caused by high protein is very large, the frequency change caused by glucose is covered, and the high non-specificity of protein (the total amount of protein in saliva can reach thousands of milligrams per liter) generates a huge background signal, so that the test is inaccurate.
Therefore, in order to solve the problems in the prior art, there is a need for a method of manufacturing a chip having an anti-fouling microgel, a chip, and a sensor.
Disclosure of Invention
One aspect of the present invention is to provide a method of manufacturing a microgel chip having stain resistance, the method comprising:
synthesis of microgel: 516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350uL of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide and 5mg of azobisisobutyronitrile are added into 40mL of acetonitrile, polymerized for 1 hour at 90 ℃, and repeatedly washed by distilled water to obtain microgel;
synthetic microgel antifouling layer: threonine and serine in a molar ratio of 1:1, adding the mixture into an ethanol solution, regulating the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of microgel into the mixed solution, reacting for 24 hours at 50 ℃, washing the product with distilled water to be neutral, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance;
is fixed on the crystal oscillator piece: 30mg of the white paste was taken, 50. Mu.L of ethylene glycol dimethacrylate, 5. Mu.L of 2, 2-dimethoxy-phenyl ethanone was dispersed in 50. Mu.L of dimethyl sulfoxide to prepare a prepolymerized solution,
and then 25 mu L of the pre-polymerized solution is placed on a quartz plate, the front surface of a crystal oscillator piece is placed on the pre-polymerized solution in a downward manner, the back surface of the crystal oscillator piece is pressed by a film pressing machine, an ultraviolet lamp irradiates for 1 hour, distilled water is flushed, and nitrogen is used for drying.
Preferably, the method further comprises the following treatment of the crystal oscillator wafer before the microgel antifouling layer is fixed on the crystal oscillator wafer:
ultrasonic treating the crystal oscillator sheet in piranha solution for 10 min, flushing the crystal oscillator sheet with distilled water and blow-drying with nitrogen;
then put into ethanol containing 100 mu L of gamma-aminopropyl triethoxysilane to react for 12 hours at room temperature;
the crystal oscillator sheet is washed by distilled water and dried by nitrogen, then N, N-dimethylformamide of maleic anhydride is put into the crystal oscillator sheet to react for 12 hours, and the crystal oscillator sheet is washed by distilled water again and dried by nitrogen.
Preferably, the piranha solution is: h with mass percentage concentration of 96% w/w 2 SO 4 And H with mass percentage concentration of 30% w/w 2 O 2 According to the volume ratio of 7:3, and the obtained mixed solution.
Preferably, threonine and serine are present in a molar ratio of 1:1 into an ethanol solution, wherein the volume ratio of ethanol to water of the ethanol solution is 1:3.
another aspect of the present invention is to provide a chip including a crystal oscillator sheet to which an antifouling microgel is immobilized by chemical crosslinking;
the antifouling microgel is formed into a microgel antifouling layer by grafting amino acid to the microgel, so that the nonspecific adsorption of protein is reduced for the chip.
Preferably, the crystal oscillator plate fixes the antifouling microgel by chemical crosslinking as follows:
the crystal oscillator wafer is processed as follows:
ultrasonic treating the crystal oscillator sheet in piranha solution for 10 min, flushing the crystal oscillator sheet with distilled water and blow-drying with nitrogen;
then put into ethanol containing 100 mu L of gamma-aminopropyl triethoxysilane to react for 12 hours at room temperature;
washing the crystal oscillator sheet with distilled water, drying with nitrogen, adding N, N-dimethylformamide of maleic anhydride, reacting for 12 hours, washing with distilled water again, and drying with nitrogen;
fixing the antifouling microgel on the crystal oscillator plate through chemical crosslinking:
30mg of the microgel anti-fouling layer, 50 mu LN-vinyl pyrrolidone, 5 mu L of ethylene glycol dimethacrylate and 3mg of 2, 2-dimethoxy-phenyl ethanone are dispersed in 50 mu L of dimethyl sulfoxide to prepare a pre-polymerized solution,
and then 25 mu L of the pre-polymerized solution is placed on a quartz plate, the front surface of a crystal oscillator piece is placed on the pre-polymerized solution in a downward manner, the back surface of the crystal oscillator piece is pressed by a film pressing machine, an ultraviolet lamp irradiates for 1 hour, distilled water is flushed, and nitrogen is used for drying.
Preferably, the piranha solution is: h with mass percentage concentration of 96% w/w 2 SO 4 And H with mass percentage concentration of 30% w/w 2 O 2 According to the volume ratio of 7:3, and the obtained mixed solution.
Preferably, the microgel is synthesized by the following method:
516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350uL of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide, 5mg of azobisisobutyronitrile were added to 40mL of acetonitrile, polymerized at 90℃for 1 hour, and repeatedly washed with distilled water to obtain microgel.
Preferably, the microgel anti-fouling layer is synthesized by the following method:
threonine and serine in a molar ratio of 1:1, adding the mixture into an ethanol solution, regulating the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of microgel into the mixed solution, reacting for 24 hours at 50 ℃, washing the product with distilled water to be neutral, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance.
A further aspect of the invention is to provide a sensor comprising the claim chip, the chip comprising a crystal oscillator wafer, the crystal oscillator wafer fixing the anti-fouling microgel by chemical crosslinking;
the antifouling microgel is formed into a microgel antifouling layer by grafting amino acid to the microgel, so that the nonspecific adsorption of protein is reduced for the chip.
The preparation method of the chip with the antifouling microgel, the chip and the sensor provided by the invention synthesize the antifouling microgel on the QCM chip, and overcome the non-specific adsorption of protein when detecting saliva glucose.
The preparation method of the chip with the antifouling microgel, the chip and the sensor provided by the invention have the advantages that the microgel containing boric acid is used as a binding site of glucose, amino acid grafted on the microgel is used as a protein resistant component, and then the amino acid is fixed on the chip through chemical crosslinking, so that the glucose sensor has glucose responsiveness and pollution resistance, and can detect 0-40 mg/L salivary glucose under physiological conditions.
The preparation method of the anti-fouling microgel chip, the chip and the sensor provided by the invention have excellent protein and organic molecule tolerance capability, and realize the detection (V) of glucose in saliva solution Saliva ＝V PBS ＝1:1)。
The preparation method of the anti-fouling microgel chip, the chip and the sensor provided by the invention can eliminate the problem of inaccurate test caused by high non-specificity of protein under the condition that the content of glucose molecules in saliva is extremely low (3.6-36 mg/L) and the frequency change is very small.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
Drawings
Further objects, functions and advantages of the present invention will be clarified by the following description of embodiments of the present invention with reference to the accompanying drawings, in which:
FIG. 1 schematically shows a flow chart of the preparation of a chip with an anti-fouling microgel in one embodiment of the invention.
Figure 2a shows the synthesized microgel microstructure.
Fig. 2b shows the microscopy of the synthetic microgel anti-fouling layer.
Fig. 2c shows the microstructure after the fixation of the microgel anti-fouling layer on the chip.
FIG. 3 shows a stability test pattern for a microgel chip with stain resistance according to the present invention.
FIG. 4 shows a schematic diagram of the detection limit test of the present invention with an anti-fouling microgel chip.
FIG. 5 shows a schematic representation of the effect of different pHs on the test with an anti-fouling microgel chip according to the invention.
FIG. 6 shows a schematic diagram of a reversion test with an anti-fouling microgel chip according to the invention.
FIG. 7 shows a schematic of a real saliva test with an anti-fouling microgel chip of the invention.
Detailed Description
The objects and functions of the present invention and methods for achieving these objects and functions will be elucidated by referring to exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; this may be implemented in different forms. The essence of the description is merely to aid one skilled in the relevant art in comprehensively understanding the specific details of the invention.
Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the drawings, the same reference numerals represent the same or similar components, or the same or similar steps.
In order to solve the problem that the protein in the prior art generates a huge background signal with high non-specificity, which leads to inaccuracy in saliva sugar detection, the preparation method of the anti-fouling microgel chip is provided.
Embodiment one.
In one embodiment of the present invention, a flowchart of a method for preparing a chip with an anti-fouling micro-gel, as shown in fig. 1, includes:
before the microgel antifouling layer is fixed on the crystal oscillator wafer, the crystal oscillator wafer is treated
The wafer was sonicated in piranha solution for 10 minutes, distilled water rinsed wafer 1 and blow dried with nitrogen.
Then, the mixture was put into ethanol (50 mL) containing 100. Mu.L of gamma-aminopropyl triethoxysilane and reacted at room temperature for 12 hours.
The wafer was rinsed with distilled water and dried with nitrogen, and then N, N-dimethylformamide (50 mL) was added with maleic anhydride (1 g) to react for 12 hours, and again rinsed with distilled water and dried with nitrogen.
piranha solution was: h with mass percentage concentration of 96% w/w 2 SO 4 And H with mass percentage concentration of 30% w/w 2 O 2 According to the volume ratio of 7:3, and the obtained mixed solution.
Synthetic microgel
516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350uL of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide and 5mg of azobisisobutyronitrile are added into 40mL of acetonitrile, polymerized for 1 hour at 90 ℃, and repeatedly washed by distilled water to obtain microgel;
synthetic microgel anti-fouling layer
Threonine and serine in a molar ratio of 1:1, adding the mixture into an ethanol solution, regulating the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of microgel into the mixed solution, reacting for 24 hours at 50 ℃, washing the product with distilled water to be neutral, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance.
In some preferred embodiments, threonine and serine are present in a molar ratio of 1:1 into an ethanol solution, wherein the volume ratio of ethanol to water of the ethanol solution is 1:3.
is fixed on the crystal oscillator plate
30mg of the white paste material (microgel anti-fouling layer), 50. Mu.L of ethylene glycol dimethacrylate, 5. Mu.L of 2, 2-dimethoxy-phenyl ethanone was dispersed in 50. Mu.L of dimethyl sulfoxide to prepare a pre-polymerization solution,
then 25 mu L of the pre-polymerized solution is placed on a quartz plate (10 multiplied by 10 cm), the front surface of a crystal oscillator sheet 1 is placed on the pre-polymerized solution, the back surface of the crystal oscillator sheet is pressed by a film pressing machine, an ultraviolet lamp (365 nm) is irradiated for 1 hour, distilled water is used for flushing, and nitrogen is used for drying, so that the microgel anti-fouling layer 2 is crosslinked and fixed on the crystal oscillator sheet 1.
And (2) implementing the second step.
The embodiment provides a chip, the chip includes a crystal oscillator wafer, the crystal oscillator wafer fixes antifouling microgel through chemical crosslinking, wherein, antifouling microgel forms a microgel antifouling layer through amino acid grafting to microgel, so that nonspecific adsorption of protein is reduced for the chip.
The crystal oscillator sheet is fixed with the antifouling microgel through the following chemical crosslinking:
the crystal oscillator wafer is processed as follows:
the wafer was sonicated in piranha solution for 10 minutes, distilled water rinsed the wafer and blow dried with nitrogen. piranha solution was: h with mass percentage concentration of 96% w/w 2 SO 4 And H with mass percentage concentration of 30% w/w 2 O 2 According to the volume ratio of 7:3, and the obtained mixed solution.
Then, the mixture was put into ethanol (50 mL) containing 100. Mu.L of gamma-aminopropyl triethoxysilane and reacted at room temperature for 12 hours.
The wafer was rinsed with distilled water and dried with nitrogen, and then N, N-dimethylformamide (50 mL) was added with maleic anhydride (1 g) to react for 12 hours, and again rinsed with distilled water and dried with nitrogen.
Fixing the antifouling microgel on the crystal oscillator plate through chemical crosslinking:
30mg of the microgel anti-fouling layer, 50 mu LN-vinylpyrrolidone, 5 mu L of ethylene glycol dimethacrylate and 3mg of 2, 2-dimethoxy-phenylethanone were dispersed in 50 mu L of dimethyl sulfoxide to prepare a prepolymerization solution.
Then 25 mu L of the pre-polymerized solution is placed on a quartz plate (10 multiplied by 10 cm), the front surface of a crystal oscillator wafer is placed on the pre-polymerized solution, the back surface of the crystal oscillator wafer is pressed by a film pressing machine, an ultraviolet (365 nm) lamp irradiates for 1 hour, distilled water is used for flushing, and nitrogen is used for drying.
Microgels are synthesized by the following method:
516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350uL of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide, 5mg of azobisisobutyronitrile were added to 40mL of acetonitrile, polymerized at 90℃for 1 hour, and repeatedly washed with distilled water to obtain microgel.
The microgel antifouling layer is synthesized by the following method:
threonine and serine in a molar ratio of 1:1, adding the mixture into an ethanol solution, regulating the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of microgel into the mixed solution, reacting for 24 hours at 50 ℃, washing the product with distilled water to be neutral, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance.
Embodiment three.
The embodiment provides a sensor, and the sensor comprises a chip, wherein the chip comprises a crystal oscillator sheet, and the crystal oscillator sheet is used for fixing antifouling microgel through chemical crosslinking, wherein the antifouling microgel is used for grafting amino acid to the microgel to form a microgel antifouling layer, so that the nonspecific adsorption of protein is reduced for the chip.
The process of crosslinking and fixing the antifouling microgel by the crystal oscillator is the same as that of the first and second embodiments, and will not be repeated here.
The preparation method of the anti-fouling microgel chip, the chip and the sensor provided by the invention are subjected to experimental verification.
1. Characterization:
scanning electron microscopy was used to evaluate the morphology and particle size of the prepared antifouling microgel.
The synthesized microgel microstructure shown in FIG. 2a has smooth surface, uniform size (about 600 nm) and spherical morphology.
The microstructure of the synthetic microgel anti-fouling layer shown in fig. 2b shows that the amino acid is grafted to the surface of the microgel after grafting, and the surface is uneven.
The microstructure of the chip after the microgel anti-fouling layer was fixed on the chip as shown in fig. 2c, and the crosslinked layer in many small ball packages demonstrated that the microgel anti-fouling layer was successfully fixed to the surface of the chip wafer.
2. Stability:
the stability of the sensor was evaluated in PBS (phosphate buffered saline, phosphate buffer saline, PBS).
The invention has a stability test pattern of an antifouling microgel chip as shown in fig. 3. When the polymer coated chip was exposed to PBS for about 2 hours, the fluctuation value of Δf (frequency shift) was only Δf= -2.4 Hz.
3. Detection limit:
in order to obtain the detection limit of the glucose sensor, the glucose concentration is gradually increased in the examples.
As shown in FIG. 4, the detection limit of the anti-fouling microgel chip provided by the invention is 5 milligrams per liter.
4. Detection range:
different ranges of glucose detection are achieved by adjusting different pH values.
As shown in FIG. 5, the invention has a schematic diagram of the effect of different pHs of the anti-fouling microgel chip on the test, and the absolute value of DeltaF gradually increases with the rise of the pH. The sensor showed good linear relationship with glucose concentrations ranging from 0 to 40 milligrams per liter, with linear correlation coefficients 0.9523 (pH 6.8), 0.9274 (pH 7.2), 0.9482 (pH 7.5), respectively
5. Recovery:
as shown in FIG. 6, the invention has a schematic diagram of the reversion test of an antifouling microgel chip, and the frequency change is basically unchanged when the chip is alternately exposed with 200 mg/L of glucose and 0mg/L of glucose for a plurality of times.
6. True saliva test:
to further demonstrate the anti-contamination performance of the glucose chip (sensor) of the present invention, real saliva detection was used in the examples.
Putting the cotton swab into the mouth, chewing for 2-3 minutes to fully absorb saliva, putting the cotton swab into a saliva collecting pipe, and centrifuging to obtain saliva. Equal amounts of saliva and PBS were mixed and different amounts of glucose were added. The invention is shown in FIG. 7 as a schematic diagram of a real saliva test with an anti-fouling microgel chip, with an increasing absolute value of ΔF (a in FIG. 7) with increasing glucose concentration. Glucose concentration has a good linear relationship with Δf, with a linear correlation coefficient of 0.9671 (b in fig. 7).
The preparation method of the chip with the antifouling microgel, the chip and the sensor provided by the invention synthesize the antifouling microgel on the QCM chip, and overcome the non-specific adsorption of protein when detecting saliva glucose.
The preparation method of the chip with the antifouling microgel, the chip and the sensor provided by the invention have the advantages that the microgel containing boric acid is used as a binding site of glucose, amino acid grafted on the microgel is used as a protein resistant component, and then the amino acid is fixed on the chip through chemical crosslinking, so that the glucose sensor has glucose responsiveness and pollution resistance, and can detect 0-40 mg/L salivary glucose under physiological conditions.
The preparation method of the anti-fouling microgel chip, the chip and the sensor provided by the invention have excellent protein and organic molecule tolerance capability, and realize the detection (V) of glucose in saliva solution Saliva ＝V PBS ＝1:1)。
The preparation method of the anti-fouling microgel chip, the chip and the sensor provided by the invention can eliminate the problem of inaccurate test caused by high non-specificity of protein under the condition that the content of glucose molecules in saliva is extremely low (3.6-36 mg/L) and the frequency change is very small.
Other embodiments of the invention will be apparent to and understood by those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Claims (6)
1. A method for preparing a microgel chip with stain resistance, comprising the steps of:
synthesis of microgel: 516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350 mu L of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide, 5mg of azobisisobutyronitrile are added into 40mL of acetonitrile, polymerization is carried out for 1 hour at 90 ℃, and distilled water is repeatedly washed to obtain microgel;
synthetic microgel antifouling layer: threonine and serine in a molar ratio of 1:1, adding the mixture into an ethanol solution, regulating the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of microgel into the mixed solution, reacting for 24 hours at 50 ℃, washing the product with distilled water to be neutral, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance;
is fixed on the crystal oscillator piece: 30mg of the white paste was taken, 50. Mu.L of ethylene glycol dimethacrylate, 5. Mu.L of 2, 2-dimethoxy-phenyl ethanone was dispersed in 50. Mu.L of dimethyl sulfoxide to prepare a prepolymerized solution,
and then placing 25 mu L of the pre-polymerized solution on a quartz plate, placing the crystal oscillator piece with the front face downwards on the pre-polymerized solution, pressing the back face of the crystal oscillator piece by using a film pressing machine, irradiating for 1 hour by using an ultraviolet lamp, flushing with distilled water and drying by nitrogen.
2. The method of claim 1, further comprising the step of treating the wafer prior to the step of affixing the microgel anti-fouling layer to the wafer:
ultrasonic treating the crystal oscillator sheet in piranha solution for 10 min, flushing the crystal oscillator sheet with distilled water and blow-drying with nitrogen;
then put into ethanol containing 100 mu L of gamma-aminopropyl triethoxysilane to react for 12 hours at room temperature;
the crystal oscillator sheet is washed by distilled water and dried by nitrogen, then N, N-dimethylformamide of maleic anhydride is put into the crystal oscillator sheet to react for 12 hours, and the crystal oscillator sheet is washed by distilled water again and dried by nitrogen.
3. The method of claim 2, wherein the piranha solution is: h with mass percentage concentration of 96% w/w 2 SO 4 And H with mass percentage concentration of 30% w/w 2 O 2 According to the volume ratio of 7:3, and the obtained mixed solution.
4. The preparation method according to claim 1, wherein threonine and serine are mixed in a molar ratio of 1:1 into an ethanol solution, wherein the volume ratio of ethanol to water of the ethanol solution is 1:3.
5. a chip, characterized in that it is prepared by the preparation method according to any one of claims 1 to 4.
6. A sensor comprising the chip of claim 5.
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